The attainment of recombinant proteins using expression vectors is a well-known fact in general, prokaryotic and yeast expression systems are highly efficacious and easy to use, whereas the expression systems used containing superior eukaryotic cells present a number of drawbacks relative to low protein production levels and limitations in the host range. Of the existing expression systems for superior eukaryotic cells, baculovirus-based vectors are the most efficacious in terms of protein production. However, they can only be used in insect cells that, as is known, glycosilate proteins differently from the way animal cells do. In addition, the construction of the recombinant virus takes place through a homologous recombination, which is a laborious technique, especially when numerous genetic variants have to be analyzed.
On the other hand, vectors based on DNA viruses suitable for heterologous gene expression are known. However, the use of DNA-based vectors presents numerous drawbacks for, as they replicate in the nucleus of the host cell and can become integrated in the genome, they are therefore not reliable. On the contrary, the use of RNA-based vectors overcomes the drawbacks associated with the use of DNA viruses because, since they replicate not in the genome of the host cell but in the cytoplasm, replication takes place via RNA and not via DNA, and the possibilities of integration in the genome are very low, making the vectors based on these RNA viruses more reliable.
Also well-known are defective interfering particles (DI) containing the virion capsid and a defective genome, which are deletion subgenomic mutants mostly generated form infectious viral genomes by a replication error. In general, the term “DI particle” refers to defective viruses lacking a region of the RNA or DNA genome, containing the proteins and antigens of the virus, requiring co-infection of the infectious parental virus (helper virus) for replication and which specifically interfere with the homologous helper virus, as they replicate at its expense [Huang and Baltimore, Nature, 226, 325-327 (1970)]. DI genomes arise from genome reorganizations as a result of shifts of the RNA polymerase from one RNA template to another or from one segment of an RNA template to another segment of the same molecule. These DI genomes, once they have been generated, self amplify at the expense of the parental genome or the amplifying virus coding for the proteins involved in replication and encapsidation and which has to compete with defective genomes for such products.
DI particles have been obtained and characterized from some coronaviruses, such as the murine hepatitis virus (MHV), infectious bronchitis virus (IBV), and bovine coronavirus (BCV), although DI particles derived from porcine transmissible gastroenteritis virus (TGEV) have not been described. One of the MHV natural DI particles has been used as the basis for the development of an expression vector in which the exogenous gene is inserted under the control of an internal promoter transcription sequence [Lin and Lai, J. Virol., 6110-6118, October. (1993)].
Generally, known heterologous gene expression vectors based on DI particles have some drawbacks related with their species and target organ specificity and their limited capacity for cloning, that limit their possibilities of use, both in basic research and in research applied to the development of such vectors for therapeutical purposes, including vaccines.
Consequently, there is still need of heterologous gene expression vectors that may successfully overcome the mentioned drawbacks. Specifically, it would be highly advantageous to have available some heterologous gene expression vectors with a high level of safety and cloning capacity and which could be designed so that their species specificity and tropism might be easily controlled.
The present invention provides a solution to the existing problem, comprising a vector based on a recombinant defective viral genome expressing antigens suitable for the induction of an immune response and for the prevention of infections caused by different infectious agents in various animal species. The heterologous gene expression vectors (or DNA sequences) provided by this invention have a high level of safety, as well as high cloning capacity, and may be designed so that their species specificity and tropism may easily be controlled, making such vectors suitable for the formulation of vaccines capable of conferring protection against infections caused by the different infectious agents in various animal species.
Therefore, an object of the present invention is a vector based on a recombinant defective viral genome expressing at least one antigen suitable for the induction of immune response—specifically, a systemic and secretory immune response against infectious agents in various animal species—, or an antibody providing protection against an infectious agent provided with a high level of safety and cloning capacity, and which may be designed so that its species specificity and tropism may be easily controlled.
The defective viral genome which serves as a basis for the construction of the said vector is also an additional objective of this invention.
Another additional object of this invention is a recombinant expression system of heterologous proteins comprising (a) the vector described above and (b) a helper virus that will provide the proteins involved in the replication and encapsidation of the recombinant defective viral genome.
Another additional objective of this invention is vaccines capable of inducing protection against infections caused by different infectious agents in various animal species, comprising the recombinant system described above, together with a pharmaceutically acceptable excipient. These vectors can be uni-, or multivalent, depending on whether the expression vectors present in the recombinant system express one, or more antigens capable of inducing an immune response against one, or more infectious agents, or one or more antibodies providing protection against one or more infectious agents.
Other objects provided by this invention comprise a method for the immunization of animals, consisting of the administration of the said recombinant system or vaccine, as well as a method for the protection of newborn animals from infectious agents that infect the mentioned species.